The objective of the proposed research is to pursue studies on glutaric acidemia type II (GA2), a human inborn error of amino and fatty acid oxidation, and on ETF:ubiquinone oxidoreductase (ETF);QO), the protein which is deficient in some patients with the disease and which normally catalyzes electron transfer between electron transfer flavoprotein (ETF) and the mitochondrial respiratory chain. Our investigations on this subject, which have been ongoing for more than ten years, have the long term objective of learning how mutations disrupt the electron transfer activity of ETF:AO, and how disease phenotype (especially renal cystic dysplasia) is caused by ETF:QO deficiency. During the last grant period we cloned cDNAs encoding human and porcine ETF:QO, identified nine mutant ETF:QO alleles in GA2 patients, expressed wild type and mutant ETF:QO in S cereviseae, crystallized porcine ETF:QO, and began to delineate mechanisms of electron transfer between the ETF:QO redox centers and ubiquinone. Specific aims for this funding period are to (a) continue to identify ETF:QO mutations that cause GA2; (b) correlate these mutations with clinical phenotype, and with functional abnormalities in expressed enzyme to develop a detailed structure-function map of ETF:QO; and, using a gene targeting approach in embryonic stem cells, (c) develop a murine model of ETF:QO deficiency, and (d) study spatial and temporal expression of ETF:QO in the developing mouse. These studies should permit better understanding of why ETF:QO deficiency so frequently causes renal cystic dysplasia and, in doing so, may permit broader understanding of the pathogenesis of other congenital anomalies as well.